IGF2BP1 Protein Binding of Erythroblast Transcription Factor mRNA: A Cytoplasm-Localized Mechanism for Globin Gene Regulation

Pharmacologic and genetic regulation of fetal hemoglobin (HbF) remains a major goal for treatment of the beta-thalassemias and sickle cell disease. Recently, the let-7 microRNA heterochronic pathway of RNA-binding factors was shown to be developmentally correlated with HbF expression in humans. Experimental manipulation of the pathway components (LIN28, let-7, and the IGF2 binding proteins) demonstrated moderate increases in fetal hemoglobin (around 15-35% HbF) with the exception of IGF2BP1, which caused a nearly complete reversal in gamma- and beta-globin gene expression resulting in HbF levels of 60-70% among cultured adult human erythrocytes. To further explore IGF2BP1 effects on HbF and erythropoiesis, lentiviral transduction of CD34(+) cells was performed with let-7 resistant expression of IGF2BP1 driven by the human SPTA1 gene (IGF2BP1-OE). Donor-matched control transductions were studied for comparison. For protein localization studies, confocal imaging of sorted cells was utilized. IGF2BP1-OE caused IGF2BP1 protein expression throughout the cytoplasm and localized to small granules. Granules were unevenly distributed and more abundantly observed in the perinuclear regions. Nuclear detection of IGF2BP1 protein remained at background control levels. We thus hypothesized that IGF2BP1 protein in these cytoplasmic granules may regulate globin gene expression by binding RNAs that encode epigenetic modifiers of the beta-globin locus. To test this hypothesis, RNA-immunoprecipitation (RIP) was performed using a RIP-certified anti-IGF2BP1 antibody. Next generation sequencing and qRT-PCR were used to determine the mRNA species bound by IGF2BP1. RIP-enrichment of BCL11A mRNA was identified (4.6 ± 1.2 fold compared to input sample), as well as mRNA encoding other modifiers of globin gene expression including KLF1 and ZBTB7A. Further analyses of BCL11A showed no significant changes in BCL11A mRNA levels among IGF2BP1-OE cells (control: 6.5.E+03 ± 3.8.E+03, IGF2BP1-OE: 4.1.E+03 ± 1.2.E+02, p=0.403). However, BCL11A protein detection was reduced to background levels by Western analysis compared to transduction control cell lysates. These studies suggest that RNA-binding and cytoplasmic compartmentalization provide IGF2BP1 with a mechanism for increasing fetal hemoglobin via post-transcriptional regulation of globin gene transcription factors.